This disclosure relates to minor groove binder (“MGB”) phosphoramidites, their synthesis, and their use in the synthesis of oligonucleotide conjugates. The phosphoramidites are useful, for example, for the automated synthesis of oligonucleotide conjugates on solid supports.
Oligonucleotide conjugates have been developed and applied widely in molecular biology in hybridization-based procedures as primers, probes, antagomers and the like. Use of these reagents in molecular biology procedures often requires modification to improve resistance to nuclease susceptibility, improve duplex stabilization of conjugate target duplexes, and allow detection or improve conjugate solubility. This then led to the inclusion of one or more non-isotopic labels such as fluorescent dyes, quenchers, intercalators, biotin, bead particles, enzymes, antibodies, antigens, hydrophobic compounds such as cholesterol, ionic compounds such as spermine, and the like. Additional modifications have been introduced in the oligonucleotide to the backbone to increase stability or nuclease resistance. These modifications include the introduction of locked nucleic acids, 2′-O-alkyl ribose units, methyl phosphonate- or phosphorothioate-, or phosphorodithoate-linkages and peptide nucleic acids. The naturally occurring bases in nucleic acids are routinely substituted with modified bases to tailor the properties of the oligonucleotide conjugate (U.S. Pat. No. 6,949,367). Minor groove binders (U.S. Pat. No. 5,801,155) and intercalators (U.S. Pat. No. 4,835,263) have been used to stabilize duplex formation. With widespread application of these compounds in diagnostics and therapeutics, there exists a need to improve reagents to satisfy the increasing demand for covalently attached minor groove binder oligonucleotide conjugates.
Minor groove binders can be attached to oligonucleotides using post synthesis conjugation (U.S. Pat. No. 5,955,590) or MGB-modified DNA synthesis solid supports (U.S. Pat. No. 5,801,155). Reagents and methods to synthesize minor groove binder conjugates are also described in U.S. Pat. No. 6,084,102. One example is a tetrafluorophenyl ester of the tripepeptide 3-carbornyl-1,2-dihydro-3H-pyrrolo[3,2,-e]indole-7-carboxylic acid residues (TFP-DPI3 carboxylate), which is an analog of the naturally occurring antibiotic CC-1065. TFP-DPI3 carboxy late can be used to synthesize a solid support for automated manufacturing of MGB-oligonucleotide conjugates. Alternatively, TFP-DPI3 can be used post synthetically to attach the MGB ligand.
Other minor groove binder oligonucleotide conjugates have been reported. Hoechst 33258 analogs with a bromoacetamide tether were coupled to oligonucleotides bearing alkyl thiol functionality (Wiederbolt et al., J. Am. Chem. Soc., 7055-7062 (1997). The same method was used to conjugate a DAPI analog to an oligonucleotide (O'Donnell et al., 1995). Hoechst 33258 can be tethered by a hexa(ethylene glycol) linker to a solid support through a cleavable linker. This solid support can then be used to synthesize the Hoechst 33258 oligonucleotide conjugate (Rajur et al., The Journal of Organic Chemistry 62: 523-529 (1997). Rajur and co-workers attempted to synthesize a Hoechst 33258-hexa(ethylene glycol) phosphoramidite but reported no success, suggesting that the benzimidazole groups of the minor groove binder competed with hydroxyl group for the phosphitylating reagent or, perhaps, were involved in other side reactions.
For some minor groove binders, phosphoramidite preparation may present challenges not only due to the presence of unwanted reactions, but also because of their very low solubility in oligonucleotide synthesis solvents such as acetonitrile. Solubility, for example, is a very pronounced problem (Boger et al., J. Org. Chem., 1521-1530 (1987)) for dihydropyrroloindole (“DPI”)-containing MGB agents belonging to the CC-1065 and duocarmycin family.
A need exists for minor groove binder phosphoramidites in the synthesis of oligonucleotide conjugates that will eliminate laborious post-synthesis attachment and, therefore, facilitate automation and scale-up. In order to achieve this goal, a need exists for an MGB protecting strategy that would be compatible with automated oligonucleotide synthesis, eliminate side reactions related to the presence of heteroaromatic amines and improve solubility in suitable organic solvents.